The overall goal of this proposal is to test our current hypotheses concerning the catalytic function of ALDH2 and the roles that specific residues play in these structure/function relationships. During the prior funding period of this award we were able to establish a unique redox-dependent coenzyme isomerization event during catalysis, as well as the mechanism by which divalent metal cations activate ALDH2. In addition, we have solved the structure of the "inactive" ALDH2*2 homotetramer both in the absence and presence of coenzyme. These studies have shown us that residue 487, which is mutated from glutamate to lysine in ALDH2*2, contributes significantly to the conformational stability of the coenzyme binding site and active site structure. In the future we propose to extend this work toward an understanding of individual catalytic steps during catalysis through the use of stopped-flow kinetics and time-dependent structural studies, including Laue crystallography. Our previous work has demonstrated that our crystals of ALDH2 are catalytically active and undergo only minor adjustments during coenzyme binding and catalysis, making Laue crystallographic experiments feasible. In a new direction, we propose to investigate the mechanistic aspects of nitroglycerin activation by ALDH2 through direct structure determination of complexes between active and inactive forms of the enzyme. The information to be gained through these aims will be useful, not only for our understanding of the role that ALDH plays in alcohol metabolism and protection from alcohol abusing behavior, but also into the mechanism by which nitroglycerin is activated for its anti-anginal properties. As part of this work, we propose to determine whether the ALDH2*2 enzyme is capable of activating nitroglycerin, since knowledge that certain individuals will or will not respond to nitroglycerin administration will be of tremendous clinical benefit. [unreadable] [unreadable]